Lutein, zeaxanthin, and their metabolites are dietary xanthophyll carotenoids that are specifically concentrated in the macula lutea of the human eye where they are thought to provide protection against degenerative diseases of the retina including age-related macular degeneration (AMD) by multiple mechanisms including filtration of phototoxic blue light and antioxidant quenching of reactive oxygen species. The body of evidence supporting their protective role against AMD has been considered strong enough to justify the inclusion of 10 mg of lutein and 2 mg of zeaxanthin in the active treatment arms of the National Eye Institute's AREDS2 study. The overall goal of this ongoing research project has been to provide a solid basic science research foundation to explain how and why lutein and zeaxanthin out of over 600 carotenoids in nature and ~15 present in the bloodstream are uniquely concentrated in the macula of the human eye in order to provide rational, scientifically based guidance for nutritional interventions against AMD through these two Specific Aims: 1) Biochemical and Imaging studies of the mechanisms of action and modes of uptake of the macular carotenoids. The identification and characterization of the currently unidentified enzyme(s) responsible for the unique conversion of dietary to lutein to meso-zeaxanthin will be initiated and completed, and the potential roles played by the carotenoid cleavage enzymes BCO1 and BCO2 in regulating carotenoid levels in the mammalian eye will be explored. Capitalizing on extensive experience in constructing noninvasive instruments to measure carotenoids in tissues, a higher-resolution confocal resonance Raman imaging system will be constructed that should allow two fundamental questions of ocular carotenoid physiology to be answered. Are the macular pigment carotenoids primarily localized to photoreceptor axons or to foveal Mller cells? Are lutein and zeaxanthin differentially distributed in the macula, and if so, can different physiological roles of the two major carotenoids (e.g. light filtration versus antioxidant) be inferred? 2) Animal models of carotenoid action in the vertebrate eye. No other mammals besides humans and fellow primates are known to take up carotenoids into ocular tissues at more than trace amounts. In the upcoming grant period, carotenoid uptake into rodent tissues will be optimized diets through dietary adjuvants, upregulation of intestinal uptake by moderate vitamin A deficiency, and cross breeding of various transgenic mice targeted to the carotenoid metabolic pathways. Results of these experiments will provide insights into the unique evolution of the macula lutea of the retina and will provide the research community with well characterized mouse pharmacokinetic and pharmacodynamic data of how much lutein and zeaxanthin can be delivered to the rodent retina and other tissues under optimal conditions. This work will facilitate mouse model studies of the physiological role of lutein and zeaxanthin in protection against ocular pathology.